1. Field of the Invention
The present invention relates to the fields of microbiology, molecular biology, cell biology and biochemistry. More specifically, the present invention relates to manipulating reductive metabolic processes in vivo using genetic and metabolic engineering, thereby allowing external control of intracellular nicotinamide adenine dinucleotide (NADH) availability. Further, the present invention relates to a method of producing increased reduced metabolites such as ethanol through aerobic or anaerobic growth of a living system comprised of a recombinant NADH recycling system.
2. Related Art
The metabolic pathways leading to the production of most industrially important compounds involve oxidation-reduction (redox) reactions. Biosynthetic transformations involving redox reactions offer a significant economic and environmental advantage for the production of fine chemicals over conventional chemical processes, in particular those redox reactions requiring stereospecificity. Furthermore, biodegradation of toxic chemicals often also involves redox reactions.
Nicotinamide adenine dinucleotide (NAD) functions as a cofactor in over 300 redox reactions and regulates various enzymes and genetic processes (Foster et al., 1990). The NADH/NAD+ cofactor pair plays a major role in microbial catabolism in which a carbon source, such as glucose, is oxidized using NAD+ producing reducing equivalents in the form of NADH. It is crucially important for continued cell growth that this reduced NADH be oxidized to NAD+ and a redox balance be achieved. Under aerobic growth, oxygen achieves this recycling by acting as the oxidizing agent. While under anaerobic growth, and in the absence of an alternate oxidizing agent, the regeneration of NAD+ is achieved through fermentation by using NADH to reduce metabolic intermediates.
The metabolic pathways leading to the production of most industrially important compounds involve redox reactions. Biosynthetic transformations involving redox reactions also offer a considerable potential for the production of fine chemicals over conventional chemical processes, especially those requiring stereospecificity.
Enzymes referred to in general as oxidoreductases, or more specifically as oxidases, reductases or dehydrogenases, catalyze these biological redox reactions. These enzymes require a donor and/or an acceptor of reducing equivalents in the form of electrons, hydrogen or oxygen atoms. Cofactor pairs that are transformed reversibly between their reduced and oxidized states, nucleotide cofactors such as NADH/NAD+ and NADPH/NADP+ among others, serve as donors and/or acceptors of reducing equivalents very effectively in a living cell.
The NADH/NAD+ cofactor pair has demonstrated a regulatory effect on gene expression and enzymatic activity. Examples include, among others, the induction by NADH of adhE expression, which encodes an alcohol dehydrogenase (Leonardo et al., 1993; Leonardo et al., 1996) and catalyzes the production of ethanol during fermentation, the inhibition by high NADH/NAD+ ratios on the pyruvate dehydrogenase complex (Graef et al., 1999), and the regulation by the NADH/NAD+ ratio on the shift between oxidation or reduction of L-lactaldehyde (Baldoma and Aguilar, 1988).
The ratio of the reduced to oxidized form of this cofactor, the NADH/NAD+ ratio, is critical for the cell. The NAD(H/+) cofactor pair is very important in microbial catabolism, where a carbon source, such as glucose, is oxidized through a series of reactions utilizing NAD+ as a cofactor and producing reducing equivalents in the form of NADH. It is crucially important for the continued growth of the cell that this reduced NADH be oxidized to NAD+, thus achieving a redox balance. Under aerobic growth, oxygen achieves this by acting as the oxidizing agent. While under anaerobic growth and in the absence of an alternate oxidizing agent, this process occurs through fermentation, where NADH is used to reduce metabolic intermediates and regenerate NAD+ (FIG. 1).
The high influence of cofactors in metabolic networks has been evidenced by studies in which the NADH/NAD+ ratio has been altered by feeding carbon sources possessing different oxidation states (Alam and Clark, 1989; Leonardo et al., 1996), by supplementing anaerobic growth with different electron acceptors, such as fumarate and nitrate (Graef et al., 1999) and by expressing an enzyme like NADH oxidase (Lopez de Felipe et al., 1998). Other previous efforts to manipulate NADH levels have included the addition of electron dye carriers (Park and Zeikus, 1999) and the variation of oxidoreduction potential conditions (Riondet et al., 2000).
The effective regeneration of used cofactors is critical in industrial cofactor-dependent production systems because of the impeding high cost of cofactors such as NAD. The cofactors, also referred to as co-enzymes, NAD+ and NADP+ are expensive chemicals, thereby making their regeneration by reoxidation to the original state imperative if they are to be used economically in low cost, chemical production systems. Efforts to do such have been described. U.S. Pat. No. 4,766,071 describes in vitro regeneration of NADH using a cell lysate of Clostriduim kluyveri as a biocatalyst and an aldehyde as an oxidizing agent. U.S. Pat. No. 5,393,615 describes electrochemical regeneration of NADH using an electrode characterized by a mediator function. Similarly, U.S. Pat. No. 5,264,092 discloses mediators covalently attached to a polymeric backbone wherein the polymeric backbone coats the surface of an electrode. U.S. Pat. No. 5,302,520 discloses a NAD regeneration system and an adenosine phosphate regeneration system that, in the presence of pyruvate, yields a labeled carbohydrate.
In enzyme bioreactors, NAD+-dependent formate dehydrogenase (FDH) from methylotrophic yeast and bacteria is extensively used to regenerate NADH from NAD+ in vitro. FDH catalyzes the practically irreversible oxidation of formate to CO2 and the simultaneous reduction of NAD+ to NADH. This system of cofactor regeneration has been successfully applied in the production of optically active amino acids (Galkin et al., 1997), chiral hydroxy acids, esters, alcohols, and other fine chemicals synthesized by different dehydrogenases (Hummel and Kula, 1989), (Tishkov et al., 1999). Purified FDH has also been used to regenerate NADH in vitro for the industrial production of non-natural amino acids that cannot be obtained by fermentation, such as L-tert-leucine which has important applications when used in pharmaceuticals (Kragl et al., 1996).
In spite of these advances, biotransformation with whole cells remains the preferred industrial method for the synthesis of most cofactor-dependent products. In these systems, the cell naturally regenerates the cofactor; however, the enzyme of interest has to compete for the required cofactor with a large number of other enzymes within the cell. For this reason, in cofactor-dependent production systems utilizing whole cells, after the enzymes of interest have been overexpressed, cofactor levels and the availability of the required form of the cofactor (reduced or oxidized) become crucial for optimal production.
Furthermore, one of the long-sought goals in recombinant polypeptide production processes is to achieve a high cloned gene expression level and high cell density. Unfortunately, under these demanding conditions, the amount of acetate accumulated in the reactor increases precipitously. Acetate accumulation is associated with decreased recombinant polypeptide productivity (Aristidou et al, 1995). Methods of controlling acetate production would be beneficial in increasing recombinant polypeptide yield in large-scale industrial synthesis of polypeptides. Additionally, the sort of metabolic manipulation used to increase recombinant polypeptide yields could also be applied to the production of any biomolecule in a large-scale system in which the stress of biomolecule production normally leads to acetate accumulation, such as biopolymers.
Catalytic hydrodesulfurization has the potential to remove sulfur from various fuels. However, this technology is associated with high costs due to hydrogen consumption and heavy metal deactivation of the catalyst. A lower cost treatment is microbiological biodesulfurization. U.S. Pat. No. 6,337,204 describes a Rhodococcus bacterial culture capable of biodesulfurization. One obstacle in this method is that these reactions require NADH as a cofactor, the availability of which is a limiting factor.
Although it is generally known that cofactors play a major role in the production of different fermentation products, their role has not been studied thoroughly and systematically in engineered systems. Instead, metabolic engineering studies have focused on manipulating enzyme levels through the amplification, addition or deletion of a particular pathway. Such steps relegate cofactor manipulations as a powerful tool for metabolic engineering, as many enzymes require them. The dehydrogenases are but one example of selective catalysis requiring the energy-transferring redox couple, NADH/NAD+.
Prior to the present invention, a genetic means of manipulating the availability of intracellular NADH in vivo by regenerating NADH through the heterologous expression of an NAD+-dependent formate dehydrogenase was not known. By way of the present invention, the effect of manipulating intracellular NADH on the metabolic patterns in Escherichia coli under anaerobic and aerobic conditions by substituting the native cofactor-independent formate dehydrogenase (FDH) by an NAD+-dependent FDH such as from Candida boidinii is described. This manipulation provoked a significant change in the final metabolite concentration pattern both anaerobically and aerobically. Under anaerobic conditions, the production of more reduced metabolites was favored, as evidenced by a dramatic increase in the ethanol to acetate ratio. Unexpectedly during aerobic growth, the increased availability of NADH induced a shift to fermentation even in the presence of oxygen by stimulating pathways that are normally inactive under these conditions.